Human embryonic stem cells (hESCs) have the unconstrained capacity for long-term stable undifferentiated growth in culture and the intrinsic potential for differentiation into all somatic cell types in the human body [1, 2]. Derivation of hESCs, essentially the in vitro representation of the pluripotent inner cell mass (ICM) or epiblast of the human blastocyst, provides not only a powerful in vitro model system for understanding the human embryonic development, but also a pluripotent reservoir for in vitro derivation of a large supply of disease-targeted human somatic cells that are restricted to the lineage in need of repair [1, 2]. However, how to channel the wide differentiation potential of human pluripotent cells efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, which yields mixed populations of cell types that may reside in three embryonic germ layers and often makes desired differentiation not only inefficient, but uncontrollable and unreliable as well [1, 2]. Although such cells can differentiate spontaneously in vitro into cells of all germ layers by going through a multi-lineage aggregate or embryoid body stage, only a small fraction of cells pursue a given lineage. In those hESC-derived multi-lineage aggregates or embryoid bodies, the simultaneous appearance of a substantial amount of widely divergent undesired cell types that may reside in three embryonic germ layers often makes the emergence of desired phenotypes not only inefficient, but uncontrollable and unreliable as well. Following transplantation, these pluripotent-cell-derived grafts tend to display not only a low efficiency in generating the desired cell types necessary for reconstruction of the damaged structure, but also phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity [1, 2]. Currently, the first-generation of hESC-derived cellular products contains variable levels of mixed populations of cell types, including residual undifferentiated hESCs and partially differentiated cells that retain the capacity to proliferate and differentiate into unwanted cells, raising a potential safety concern. In view of growing interest in the use of human pluripotent cells, including artificially-reprogrammed human induced pluripotent stem cells (hiPS cells) from non-embryonic or adult cell sources, teratoma formation and the emergence of inappropriate cell types have become a constant concern following transplantation [1, 2]. Without a practical strategy to convert pluripotent cells direct into a specific lineage, previous studies and profiling of pluripotent hESCs and their differentiating multi-lineage aggregates have provided little implications to molecular controls in human embryonic development. Developing a novel practical approach that permits to channel the wide differentiation potential of human pluripotent cells efficiently and predictably to a desired phenotype is not only vital to harnessing the power of hESC biology for safe and effective cell-based therapies, but also crucial for unveiling the molecular and cellular cues that direct human embryogenesis.
The hESC lines initially were derived and maintained in co-culture with growth-arrested mouse embryonic fibroblasts (MEFs) [1]. Although several human feeder, feeder-free, and chemically-formulated culture systems have been developed for hESCs, the elements necessary and sufficient for sustaining the self-renewal of human pluripotent cells remain unsolved [1]. These exogenous feeder cells and biological reagents help maintain the long-term stable growth of undifferentiated hESCs whereas mask the ability of pluripotent cells to respond to developmental signals. Therefore, a defined culture system for maintenance of hESCs might not only render specification of clinically-relevant early lineages directly from the pluripotent state without an intervening multi-lineage germ-layer or embryoid body stage, but also allow identify the signaling molecules necessary and sufficient for inducing the cascade of organogenesis in a process that may emulate the human embryonic development [1].
Current therapeutic approaches for a wide range of neurological diseases and injuries provide symptomatic relief but none of them change the prognosis of disease. Therefore, there is a large unfulfilled need for cell-based therapies to provide regeneration and replacement options to restore the lost nerve tissue and function. However, to date, lacking of a clinically-suitable source of engraftable human stem/progenitor cells with adequate neurogenic potential has been the major setback in developing safe and effective cell-based therapies for restoring the damaged or lost central nervous system (CNS) structure and circuitry in a wide range of neurological disorders. The traditional sources of engraftable human stem cells with neural potential for transplantation therapies have been multipotent human neural stem cells (hNSCs) isolated directly from the CNS [3]. These CNS-derived primary hNSCs are neuroepithelial-like cells that are positive for nestin and can spontaneously differentiate into a mixed population of cells containing undifferentiated hNSCs, neurons, astrocytes, and oligodendrocytes in vitro and in vivo [3]. However, cell therapy based on CNS tissue-derived hNSCs has encountered supply restriction and difficulty to use in the clinical setting due to their declining plasticity with aging and limited expansion ability, which makes it difficult to maintain a large scale and prolonged culture and potentially restricts the tissue-derived hNSC as an adequate source for graft material in the clinical setting [3]. Despite some beneficial outcomes, CNS-derived hNSCs appeared to exert their therapeutic effect primarily by their non-neuronal progenies through producing trophic and/or neuroprotective molecules to rescue endogenous dying host neurons [1, 3]. The engrafted tissue-derived stem/progenitor cells generated a small number of neurons that were insufficient to achieve the anticipated mechanism of neuron replacement in the damaged CNS [1, 3].
The genetically stable pluripotent hESCs proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration and repair. Therefore, they have been regarded as an ideal source to provide an unlimited supply of human neuronal cell types and subtypes for restoring the damaged or lost nerve tissue and function in CNS disorders. However, realizing the developmental and therapeutic potential of hESCs has been hindered by the inefficiency and instability of generating desired cell types from pluripotent cells through multi-lineage differentiation. Although neural lineages appear at a relatively early stage in differentiation, <5% hESCs undergo spontaneous differentiation into neurons [1]. Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders [1]. And unlike mouse ESCs, treating hESC-differentiating multi-lineage aggregates—embryoid bodies (EBs)—only slightly increases the low yield of neurons [1, 2]. Under protocols presently employed in the field, these neural grafts derived from pluripotent cells through multi-lineage differentiation yielded neurons at a low prevalence following engraftment, which were not only insufficient for regeneration or reconstruction of the damaged CNS structure, but also accompanied by unacceptably high incidents of teratoma and/or neoplasm formation [1]. Similar to CNS-derived hNSCs, these hESC-derived hNSCs are neuroepithelial-like cells that are positive for nestin and can spontaneously differentiate into a mixed population of cells containing undifferentiated hNSCs, neurons, astrocytes, and oligodendrocytes in vitro and in vivo [1, 2]. Before further differentiation, those secondary hNSCs were mechanically isolated or enriched from hESC-differentiating multi-lineage aggregates or embryoid bodies. Similar to their CNS counterpart, the therapeutic effect of these hESC-derived hNSCs was mediated by neuroprotective or trophic mechanism to rescue dying host neurons, but not related to regeneration from the graft or host remyelination [1, 2]. Growing evidences indicate that these secondary hNSCs derived from hESCs via conventional multi-lineage differentiation in vitro appear to have increased risk of tumorigenicity but not improved neurogenic potential compared to primary hNSCs isolated from the CNS tissue in vivo, remaining insufficient for CNS regeneration [1, 2].
To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the damaged human myocardium, either by endogenous cells or by cell-based transplantation or cardiac tissue engineering [1, 2]. In the adult heart, the mature contracting cardiac muscle cells (cardiomyocytes) are terminally differentiated and unable to regenerate. Damaged or diseased cardiomyocytes are removed largely by macrophages and replaced by non-functional cells or scar issue. Although cell populations expressing stem/progenitor cell markers have been identified in postnatal hearts, the minuscule quantities and growing evidences indicating that they are not genuine heart cells and that they differentiate predominately to smooth muscle cells rather than functional contractile cardiomyocytes have caused skepticism if they can potentially be harnessed for cardiac repair [1, 2]. There is no evidence that stem/precursor/progenitor cells derived from other sources, such as mesenchymal stem cells, bone marrow cells, umbilical cord stem cells, cord blood cells, patients' heart tissue, placenta, or fat tissue are able to give rise to the contractile heart muscle cells following transplantation into the heart [1, 2]. Therefore, the need to regenerate or repair the damaged heart muscle (myocardium) has not been met by adult stem cell therapy, either endogenous or via cell delivery, in today's healthcare industry. Pluripotent hESCs proffer unique revenue to generate a large supply of cardiac lineage-committed cells as human myocardial grafts for cell-based therapy. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs or adequate human myocardial grafts, there is intense interest in developing hESC-based therapy for heart disease and failure [1, 2]. The hESCs and their derivatives are considerably less immunogenic than adult tissues [1, 2]. It is also possible to bank large numbers of human leukocyte antigen isotyped hESC lines so as to improve the likelihood of a close match [1, 2].
However, realizing the therapeutic potential of hESCs has been hindered by the inefficiency and instability of generating cardiac cells from pluripotent cells through multi-lineage differentiation. In hESC-differentiating multi-lineage aggregates (embryoid body), only a very small fraction of cells (˜1-4%) spontaneously differentiate into cardiomyocytes [1, 2]. Following mechanical isolation and immuno-selection, the small quantity of enriched cardiomyocytes could rescue the function of a damaged myocardium as a biological pacemaker following injection into the heart of animal models [1, 2]. Although such hESC-derived cardiomyocytes can attenuate the progression of heart failure in rodent models of acute myocardial infraction, they are insufficient to restore heart function or to alter adverse remodeling of a chronic myocardial infarction model following transplantation [1, 2].
It can therefore be seen that there is a need to develop new techniques for well-controlled efficiently channeling the wide differentiation potential of pluripotent hESCs exclusively and predictably to a large scale of neuronal lineage committed cells, which is vital to providing a large supply of clinically-suitable human neuronal therapeutic products across the spectrum of developmental stages in high purity and efficiency, and with adequate neurogenic potential for neuronal repair against neurological diseases or injuries.
It can therefore be seen that there is a need to develop new techniques for well-controlled efficiently channeling the wide differentiation potential of pluripotent hESCs exclusively and predictably to a large scale of cardiac lineage committed cells, which is vital to providing a large supply of clinically-suitable human cardiac therapeutic products across the spectrum of developmental stages in high purity and efficiency, and with adequate cardiogenic potential for myocardium repair against cardiovascular diseases.